Chen Guangping, Chen Xinrong
Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma 74078, USA.
J Biol Chem. 2003 Sep 19;278(38):36358-64. doi: 10.1074/jbc.M306045200. Epub 2003 Jul 16.
Cytosolic sulfotransferases (STs) catalyze the sulfation of hydroxyl containing compounds. Human phenol sulfotransferase (SULT1A1) is the major human ST that catalyzes the sulfation of simple phenols. Because of its broad substrate specificity and lack of endogenous substrates, the biological function of SULT1A1 is believed to be an important detoxification enzyme. In this report, amino acid modification, computer structure modeling, and site-directed mutagenesis were used for studies of Arg residues in the active site of SULT1A1. The Arg-specific modification reagent, 2,3-butanedione, inactivated SULT1A1 in an efficient, time- and concentration-dependent manner, suggesting Arg residues play an important role in the catalytic activity of SULT1A1. According to the computer model, Arg78, Arg130, and Arg257 may be important for SULT1A1 catalytic activity. Site-directed mutagenesis results demonstrated that the positive charge on Arg78 is not critical for SULT1A1 because R78A is still active. In contrast, a negative charge at this position, R78E, completely inactivated SULT1A1. Arg78 is in close proximity to the site of sulfuryl group transfer. Arg257 is located very close to the 3'-phosphate in adenosine 3'-phosphate 5'-phosphosulfate (PAPS). Site-directed mutagenesis demonstrated that Arg257 is critical for SULT1A1: both R257A and R257E are inactive. Although Arg130 is also located very close to the 3'-phosphate of PAPS, R130A and R130E are still active, suggesting that Arg130 is not a critical residue for the catalytic activity of SULT1A1. Computer modeling suggests that the ionic interaction between the positive charge on Arg257, and the negative charge on 3'-phosphate is the primary force stabilizing the specific binding of PAPS.
胞质磺基转移酶（STs）催化含羟基化合物的硫酸化反应。人酚磺基转移酶（SULT1A1）是催化简单酚类硫酸化反应的主要人源ST。由于其广泛的底物特异性以及缺乏内源性底物，SULT1A1的生物学功能被认为是一种重要的解毒酶。在本报告中，采用氨基酸修饰、计算机结构建模和定点诱变等方法研究SULT1A1活性位点中的精氨酸残基。精氨酸特异性修饰试剂2,3 - 丁二酮以高效、时间和浓度依赖性方式使SULT1A1失活，表明精氨酸残基在SULT1A1的催化活性中起重要作用。根据计算机模型，精氨酸78、精氨酸130和精氨酸257可能对SULT1A1的催化活性很重要。定点诱变结果表明，精氨酸78上的正电荷对SULT1A1并不关键，因为R78A仍然具有活性。相反，该位置的负电荷R78E则完全使SULT1A1失活。精氨酸78非常靠近硫酸基团转移位点。精氨酸257非常靠近3'-磷酸腺苷5'-磷酸硫酸酯（PAPS）中的3'-磷酸基团。定点诱变表明精氨酸257对SULT1A1至关重要：R257A和R257E均无活性。尽管精氨酸130也非常靠近PAPS的3'-磷酸基团，但R130A和R130E仍然具有活性，这表明精氨酸130不是SULT1A1催化活性的关键残基。计算机建模表明，精氨酸257上的正电荷与3'-磷酸基团上的负电荷之间的离子相互作用是稳定PAPS特异性结合的主要力量。
